Spectrophotometric determinations of transaminases (aminotransferases) present in biological fluids have been used as an aid in the diagnosis of myocardial infarction and the necrosis of hepatic cells. The enzymes of primary significance are glutamic oxalacetic transaminase, hereinafter called GOT, and glutamic pyruvic transaminase, hereafter called GPT. These enzymes catalyze the following reactions. EQU L-aspartate + .alpha.-oxoglutarate .sup.GOT oxalacetate + L-glutamate (I) EQU l-alanine + .alpha.-oxoglutarate .sup.GPT pyruvate + L-glutamate (II)
various colorimetric methods for the determination of transaminases have been described. See Reitman, et al., Amer. J. Clin. Pathol. 28, 56 (1956) and Babson, et al., Clin. Chim. Acta. 7, 199 (1962). Disadvantages associated with these methods include non-linear activity curves and long overall reaction times.
In 1970, Lippi, et al., Clin. Chim. Acta. 28, 431, described a glutamate-linked approach which utilized glutamate dehydrogenase and phenazonium methosulfate to generate a formazon dye. This method was later improved by Stavropoulos and Acuff, U.S. Pat. No. 4,024,021. In both methods, however, the presence of endogenous serum glutamate can result in falsely elevated transaminase activity.
In measuring serum GOT and serum GPT activity by the procedure of Lippi, et al. and as improved by Stavropoulos, et al., the endogenous glutamate present in the serum is converted to the final product, a formazon dye. This shows up as an initial "burst" in the product formation curve. Although the activity can be calculated from the linear portion of the product formation curve beyond the burst, if the serum level of glutamate is high the burst may be greater than two absorbance units under the conditions described hereinbelow, which makes accurate measurements difficult to obtain. In addition, the burst would result in falsely elevated activity in an end-point assay method where the reaction is stopped after a set reaction time.